DNA sequence encoding a retinoic acid regulated protein

ABSTRACT

The present invention concerns a novel retinoic acid (RA) regulated gene whose expression product displays useful morphogenic/mitogenic properties.

RELATED APPLICATIONS

[0001] This application is a Divisional of application Ser. No.09,354,359 which is incorporated herein by reference.

[0002] The present invention concerns a novel retinoic acid (RA)regulated gene whose expression product displays usefulmorphogenic/mitogenic properties.

[0003] Retinoic acid induces the differentiation of many cell types,such as epithelial cells, mesenchyme cells, teratocarcinoma cells,leukaemia cells and immortalized cell lines such as embryonal carcinomacells and neuroblastoma cells. RA is a morphogen which specifies axialpatterning during embryonic development and which affects neurogenesis,and has been used as an effective therapeutic agent for the treatment ofacute promyelocytic leukaemia.

[0004] The exact mode of action of retinoic acid is currently unknown,although it is known to be mediated by the nuclear retinoic acidreceptors (RARs) (Chambon, P., 1996, FASEB J., 10: 940-959) and it ishypothesised that the diverse effects of RA result from the differentialregulation of proteins such as transcription factors, enzymes and growthfactor receptors.

[0005] Cheung, W. M. W. et al. (1997, J. Neurochem., 68: 1882-1888) haveused RNA fingerprinting by arbitrarily primed PCR to identify a largenumber of genes that are differentially regulated during RA-inducedneuronal differentiation. The present inventors have succeeded inisolating, purifying and cloning a novel gene which is down-regulatedduring RA-induced neuronal differentiation and whose resultant proteinproduct possesses morphogenic/mitogenic properties.

[0006] According to the present invention there is provided a genehaving the sequence of SEQ ID NO: 1. Also provided is an expressionproduct encoded by the gene of the present invention, and in particularan expression product of the gene having the sequence of SEQ ID NO: 2.The present invention also extends to allelic mutants of said gene andgene expression product, and also to modified forms of said nucleic acidsequence which encode said expression product. For example,modifications may be made to the nucleic acid sequence such that it hasa different sequence yet still codes for the same amino acid sequence.

[0007] Experiments (below) show that the expression product is importantin maintaining the stem cell identity of the progenitor cells, as wellas in the early differentiation of the progenitor cells. It is alsoimportant in embryogenesis and also appears to participate in thefunctioning of adult tissues, particularly brain, lung, liver andkidney. Expression of the gene product in lymphoid tissues shows arestrictive profile in the T-cell lineage of the immune system,particularly in the thymus and the bone marrow.

[0008] The gene of the present invention may also have applications inthe treatment of Ushers disease, particularly type II Ushers disease,and thus the present invention extends to the use of the gene and itsexpression product in the manufacture of medicaments for treating Ushersdisease, together with methods of treatment of Ushers disease.

[0009] Thus the gene of the present invention is useful both in treatingand preventing diseases associated with its expression, with morphogenyand mitogeny, and with Ushers disease, particularly type II Ushersdisease.

[0010] Thus the expression product according to the present inventionmay be a mitogen and/or a morphogen.

[0011] The expression product of the present invention may be usefullyprovided in the form of a recombinant construct, allowing its expressionby chosen organisms under chosen conditions.

[0012] According to the present invention, there is also provided a DNAmolecule, which may be in recombinant or isolated form, comprising asequence encoding an expression product according to the presentinvention.

[0013] The coding sequence may be operatively linked to an expressioncontrol sequence sufficient to drive expression. Recombinant DNA inaccordance with the invention may be in the form of a vector, forexample a plasmid, cosmid or phage. A vector may include at least oneselectable marker to enable selection of cells transfected (ortransformed) with the vector. Such a marker or markers may enableselection of cells harbouring vectors incorporating heterologous DNA.The vector may contain appropriate start and stop signals. The vectormay be an expression vector having regulatory sequences to driveexpression. Vectors not having regulatory sequences may be used ascloning vectors (as may expression vectors).

[0014] Cloning vectors can be introduced into suitable hosts (forexample E. coli) which facilitate their manipulation. According toanother aspect of the invention, there is therefore provided a host celltransfected or transformed with DNA according to the present invention.Such host cells may be prokaryotic or eukaryotic. Expression hosts maybe stably transformed. Unstable and cell-free expression systems may ofcourse also be used.

[0015] Expression hosts may contain other exogenous DNA to facilitatethe expression, assembly, secretion and other aspects of thebiosynthesis of molecules of the invention.

[0016] The invention may be used with synthetic DNA sequences, cDNAs,full genomic sequences and “minigenes”, i.e. partial genomic sequencescontaining some, but not all, of the introns present in the full lengthgene.

[0017] DNA according to the present invention may be prepared by anyconvenient method involving coupling together successive nucleotides,and/or ligating oligo- and/or poly-nucleotides, including in vitroprocesses, as well as by the more usual recombinant DNA technology.

[0018] Also provided according to the present invention is a recombinantDNA construct comprising operatively linked in sequence in the 5′ to 3′direction:

[0019] a) a promoter region that directs the transcription of a gene;

[0020] b) a DNA coding sequence encoding an RNA sequence encoding anexpression product of the present invention; and

[0021] c) a 3′ non-translated region.

[0022] The DNA coding sequence may have the sequence of SEQ ID NO: 1.

[0023] Also provided is a cell transformed or transfected with arecombinant DNA construct of the present invention.

[0024] Also provided is a method of treating or preventing diseasesassociated with the expression of a gene of the present invention,comprising administering to a patient an expression product of thepresent invention.

[0025] As well as simply expressing the gene or administering the geneproduct in order to effect treatment of a patient, it may also bedesirable to inhibit (i.e. antagonise) the gene product. This can beachieved in a multitude of ways, as will be readily apparent to oneskilled in the art, and the teachings of U.S. Pat. No. 5,856,129 andreferences cited therein on how to produce and identify antagonists,inhibitors and potentiators of gene products are incorporated herein byrefernce. In particular, the following teachings may be used: Harlow, E.and Lane, D., “Using Antibodies: A Laboratory Manual”, Cold SpringHarbor Laboratory Press, New York, 1998; Sambrook, J., Frisch, E. F.,and Maniatis, T., “Molecular Cloning. A Laboratory Manual”, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, New York, 1989; Ausubel, F.M. et al., 1989, Current Protocols in Molecular Biology, John Wiley &Sons, New York, N.Y.; Gee, J. E. et al., 1994, In: Huber, B. E. andCarr, B. I. Molecular and Immunologic Approaches, Futura Publishing Co.,Mt. Kisco, N.Y.

[0026] The invention will be further apparent from the followingdescription with reference to the figures, which shows by way of exampleonly the cloning and study of the gene of the present invention.

[0027] Of the Figures:

[0028]FIG. 1 shows coupled in vitro transcription and translation usingrabbit reticulocyte extract, demonstrating that full length 8.31 cDNAencoded a ˜80 kDa protein. Histidine (His)-tagged 8.31 protein wasconstructed by cloning 6 His to the C-terminus of 8.31. Coupled in vitrotranscription and translation was performed in the absence ofradioactive label. The translated proteins were separated by SDS PAGE,transferred to nitrocellulose membrane and blotted with monoclonalantibody against the 6× His tail.

[0029]FIG. 2 shows Northern blot analysis of 8.31 expression inRA-treated NT2 cells. Total RNA (10 μg) prepared from NT2 cells treatedwith all-trans RA for 0 to 28 days, separated by denaturing gelelectrophoresis, and transferred to nylon membrane. Hybridization wasperformed using the full length 8.31 cDNA as probe. Ribosomal RNA bandsare as shown on the left.

[0030]FIG. 3 shows the expression profile of 8.31 in human tissues.Multiple Tissue Northern blots (Clontech) were hybridized using fulllength 8.31 cDNA as probe. Results of the hybridization using adulttissues (FIGS. 3A and 3B) and fetal tissues are shown (FIG. 3C). RNAsize markers are indicated on the left.

[0031]FIG. 4 shows a dot blot analysis of 8.31 expression. Messenger RNA(2 μg) was used in the dot blot to examine the expression of 8.31 invarious tissues of hematopoietic origin as well as fetal tissues.Results of the hybridization using full length 8.31 cDNA as probe areshown. Adult cells (top and middle rows) are (left to right, top tobottom) small intestine, spleen, thymus, peripheral leukocyte, lymphnode, bone marrow, trachea and placenta. Fetal cells (bottom row) are(left to right) kidney, liver, spleen thymus and lung.

[0032]FIG. 5 shows RT-PCR analysis of the 8.31 expression in human celllines. Total RNA (2 μg) obtained from neuronal precursor cell linesIMR32, and leukaemia cells was reverse transcribed and amplified byspecific primers for 8.31. KT4 represents treatment of KG1 cells withall-trans RA for 4 days. Hybridization was performed to confirm theidentity of the amplified products.

[0033]FIG. 6 shows expression of 8.31 in RA-treated HL-60 cells. TotalRNA (15 μg) from HL-60 cells treated with 0 to 6 days was used forNorthern blot analysis using full length 8.31 cDNA probe. Ribosomal RNAbands are indicated on the left.

[0034]FIG. 7 shows chromosomal localization of the gene 8.31 by FISH.Gene 8.31 was labelled and is shown marked “A”, and the specific markerfor the heterochromatin of chromosome 1 was labelled and so is shownmarked “B”.

EXPERIMENTAL

[0035] The gene of the present invention (also referred to as clone8.31) was cloned and expressed, its in vitro transcription andtranslation assayed and its chromosomal location determined. Theexpression profile of 8.31 in a range of cell types and under a range ofconditions has allowed a role for it to be determined.

Materials and Methods

[0036] Experimental methods referred to and used are standard laboratorytechniques. Where specific methods are not described or referenced, fulldescriptions and protocols are well known in the art and available inlaboratory manuals such as Harlow, E. and Lane, D., “Using Antibodies: ALaboratory Manual”, Cold Spring Harbor Laboratory Press, New York, 1998;Sambrook, J., Frisch, E. F., and Maniatis, T., “Molecular Cloning. ALaboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring HarborPress, New York, 1989; PCR (Volume 1): A practical approach. Eds. M. J.McPherson, P. Quirke and G. R. Taylor. Oxford University Press, 1991;and Torres, R. M. and Kühn, R., “Laboratory Protocols for ConditionalGene Therapy”, Oxford University Press, 1997, ISBN 019963677-X.

[0037] Cloning of full length cDNA of 8.31

[0038] Full length cDNA of 8.31 was obtained by screening an expressioncDNA library prepared from undifferentiated NT2 cells (STRATAGENE) usingthe partial 8.31 cDNA fragment (SEQ ID NO: 3) as probe. Radioactive cDNAprobes were prepared using the Megaprime DNA labelling system(AMERSHAM). Single phages were obtained and transformed into XLOLRbacterial cells (STRATAGENE) and the cDNA fragment cloned into pBK-CMVmammalian expression vector by in vivo excision.

[0039] Cell culture

[0040] NT2 cells were cultured as previously described (Cheung et al.,1996, NeuroReport, 6: 1204-1208). Cells were maintained in Opti-MEM Ireduced-serum medium (GIBCO) supplemented with 5% fetal bovine serum(FBS, GIBCO). NT2 cells were differentiated with 5 μM all-trans RA(SIGMA) in Dulbecco's modified Eagle's medium (DMEM; high glucoseformulation) supplemented with 10% FBS. Leukaemia cell lines werecultured as previously described (Xie et al., 1997, NeuroReport, 8:1067-1070).

[0041] 8.31 cDNA probe

[0042] The partial cDNA sequence of 8.31 was obtained using RNAfingerprinting by arbitrarily primed PCR (RAP-PCR, Welsh, J. et al.,1992, Nucleic Acids Res., 20: 4965-4970). Total RNA was obtained fromNT2 cells treated for various durations with all-trans RA (10 M). Thedifferentially-regulated cDNA fragments were cloned into pCRscript SK+for DNA sequencing. The cDNA probe (SEQ ID NO: 3) was then used toscreen an undifferentiated human NT2 cell cDNA library for the fulllength 8.31 cDNA.

[0043] RNA preparation, RT-PCR and Northern blot analysis

[0044] Total RNA was prepared using Trizol reagent (GIBCO) or aspreviously described (Xie et al., 1997, NeuroReport, 8: 1067-1070).Equal amounts of total RNA from different cell lines were used forNorthern blot analysis, while 2 μg total RNA was used for reversetranscription using Superscript II reverse transcritptase (GIBCO). Onetenth of the reaction was amplified using Taq DNA polymerase (GIBCO).Gene expression was confirmed by using different numbers of PCR cyclesand hybridization using 8.31 specific cDNA probes.

[0045] Coupled in vitro-transcription and translation

[0046] Two micrograms of plasmids were used for each coupled in vitrotranscription/translation reaction using the TNT coupled reticulocytelysate system (PROMEGA).

[0047] Chromosomal localization of 8.31 by FISH (fluorescent in situhybridisation)

[0048] Genomic DNA encoding 8.31 was labelled with digoxigenin (DIG)dUTP by nick translation and was hybridized to normal metaphasechromosomes derived from PHA phytohemaglutinin stimulated peripheralblood lymphocytes. After incubation with fluorescein-conjugated anti-DIGantibodies, the cells were counterstained with DAPI(4,6-diamidino-2-phenylindole), a fluorescent DNA groove-binding probe.

RESULTS

[0049] Cloning of full length coding sequence of 8.31

[0050] The cDNA encoding the full length 8.31 was obtained from a cDNAlibrary prepared from undifferentiated NT2 cells using hybridizationscreening. Double stranded sequencing by T7 DNA polymerase revealed thatthe cDNA (˜2831 bp) is novel in its gene identity (FIG. 1). The codingsequence can be translated into a protein of 730 amino acid residues.Coupled in vitro transcription and translation was performed todemonstrate that the cloned cDNA can be translated into a protein withmolecular weight of ˜80 kDa (FIG. 1).

[0051] Transcript expression of 8.31

[0052] The full length 8.31 was then used as a probe to examine itsexpression when the NT2 cells were treated with RA for 0 to 28 days(FIG. 2). Two transcripts were obtained (˜4.5 kb and ˜3.5 kb). Theexpression of 8.31 was slightly induced after 1 day of RA treatment. Atday 2, the expression decreased to its basal level and then continue todecrease along the course of RA treatment. Its expression was almosthalted at day 28.

[0053] To obtain clues on the potential functions of 8.31, we haveexamined the expression profile of 8.31 in both adult and fetal humantissues. Among the adult tissues examined, which include heart, brain,placenta, lung, liver, skeletal muscle, kidney, pancreas, stomach, andtestis, prominent expression of 8.31 was observes in placenta andtestis. Skeletal muscles expressed low levels of 8.31 (FIG. 3, panels Aand B).

[0054] The expression of 8.31 was observed in all the human fetaltissues examined, which included brain, lung, liver and kidney. An extratranscript (˜5.5 kb) was observed in all fetal tissues and a smalltranscript (˜2.4 kb) was observed only in the messenger RNA preparedfrom the fetal lung (FIG. 3, panel C). The high expression of 8.31 inthe fetal tissues examined was not observed in the corresponding adulttissues.

[0055] Dot blot analysis was performed to examine the expression of 8.31in hematopoietic tissues. Expression of 8.31 was detected in allhematopoietic tissues examined; however, 8.31 was predominantlyexpressed in the thymus and the bone marrow. Lower transcript expressionof 8.31 was detected in the spleen and lymph node. Only a barelydetectable level of its expression was observed in the peripheralleukocytes (FIG. 4).

[0056] Expression of 8.31 in different human cell lines

[0057] Owing to the high expression of 8.31 detected in thehematopoietic tissues, we have examined its expression in severalleukaemia cell lines to obtain clues on its roles in hematopoieticsystems. RT PCR analysis was performed using total RNA prepared fromK562, KG1, HL-60, HL-60S4, and CEM, cell lines each corresponding to adifferent type of leukaemia (FIG. 5). Transcript expression of 8.31 wasobserved in all hematopoietic cell lines tested. Its expression was alsoobserved in a human neuroblastoma cell line, IMR32 cells.

[0058] Expression of 8.31 was down-regulated in RA-treated HL-60 and KG1cells

[0059] HL-60 cells were differentiated with 1 μM all-trans RA and theexpression of 8.31 was examined by Northern blot analysis. Twotranscripts (˜5.5 kb and ˜3.5 kb) were detected in undifferentiatedHL-60 cells (FIG. 6). When HL-60 cells were treated with RA for 3 days,its expression was significantly down-regulated (FIG. 6). At day 6 of RAtreatment, the expression of 8.31 was diminished.

[0060] The expression of 8.31 was also down-regulated when KG1 cellswere treated with 1 μM all-trans RA, as demonstrated by the RT-PCRanalysis (FIG. 5).

[0061] Chromosomal localization of clone 8.31 by Fluorescence In SituHybridization

[0062] DNA from a genomic clone of 8.31 was labelled with digoxigenindUTP by nick translation. Labelled probe was combined with sheared humanDNA and hybridized to normal metaphase chromosomes derived from PHAstimulated peripheral blood lymphocytes. The initial experiment resultedin specific labelling of the long arm of a group A chromosome which wasbelieved to be chromosome 1 on the basis of size, morphology, andbanding pattern. A second experiment was conducted in which abiotin-labelled probe specific for the heterochromactic region ofchromosome 1 was co-hybridized with the genomic clone of 8.31. Thisexperiment resulted in a specific labelling of the heterochromatin inred (marked “B” in FIG. 7) and the long arm in green (marked “A” in FIG.7) of the chromosome 1. Measurements of 10 specifically labelledchromosomes 1 demonstrated that the genomic clone of 8.31 is located ata position which is 62% of the distance from theheterochromatic-euchromatic boundary to the telomere of chromosome arm1q, an area which corresponds to band 1q32.1-32.2 (FIG. 7). A total of80 metaphase cells were analyzed with 76 exhibiting specific labelling.

[0063] Type II Ushers syndrome (classical retinitis pigmentosa combinedwith congenital pedial deafness, and normal vestibular function) hasbeen mapped to the chromosomal region containing the gene of the presentinvention (Kimberling et al., 1990, Genomics, 2: 245-249); Lewis et al.,1990, Genomics, 2: 250-256) and it appears that the gene of the presentinvention, together with its expression product, may be useful in thetreatment of Ushers syndrome. For example, the lack of functionresulting from mutations in the diseased gene may be complemented by thegene and/or expression products of the present invention.

DISCUSSION

[0064] Functional roles of 8.31

[0065] The expression profile observed for 8.31 suggests a potentialrole in tissues of hematopoietic origin. Recently, placental blood hasbeen used as a rich source of hematopoietic stem cells fortransplantation. Taken together with the high expression of 8.31 in thetestis and the undifferentiated NT2 cells, the expression of 8.31 inplacenta revealed a strong association of the gene to the identity ofthe stem cells. Hence it appears that the gene product of 8.31 isimportant in maintaining the stem cell identity of the progenitor cells,as well as in the early differentiation of the progenitor cells.

[0066] The expression of 8.31 is also strongly associated with the earlyembryonic development. This is exemplified by the high expression of8.31 in fetal tissues such as brain, lung, liver and kidney, but not insame adult tissues. Together with its restrictive expression pattern inthe adult tissues, it appears that the gene product of 8.31 is not onlyimportant in the embryogenesis, but is also participates in thefunctioning of these adult tissues. Different 8.31 isoforms exist, theexpression of which can be regulated during the development (FIG. 3).

[0067] The predominant expession of 8.31 in the thymus and the bonemarrow, but low expression in other lymphoid tissues revealed itsrestrictive functions in the T-cell lineage of the immune system.

[0068] Involvement of 8.31 in the differentiation of cancer cells

[0069] Northern blot analysis demonstrated the down-regulation of 8.31expression with the treatment of all-trans RA. HL-60 is an acutepromyelocytic leukaemia cell line. The growth rate was sharply decreasedby treatment with RA. It appears that the expression of 8.31 is stronglyassociated with the differentiation of other cancer cell lines,including the embryonal carcinoma cells and the neuroblastoma cells.Hence 8.31 may serve as a diagnostic marker for different cancer types.

[0070] 8.31 serves as a candidate gene for genetic diseases

[0071] The gene encoding 8.31 was localized to the chromosome 1q32.132.2 Chromosome 1q 32 locus has been mapped to several genetic diseasesincluding the complement system malfunctioning, as well as the Usherdisease, which is related to hearing. Moreover the Alzheimer's diseaseis also mapped to the region 1q32, although the exact position remainsto be elucidated.

1 3 1 2831 DNA Homo sapiens CDS (124)..(2313) 1 ggcacgagcg ggagttggaggcgataacga tttgtgttgt gagaggcgca acgtgcgatt 60 tctgctgaac ttggaggcatttctacgact tttctctcag ctgaggcttt tcctccgacc 120 ctg atg ctc ttc aat tcggtg ctc cgc cag ccc cag ctt ggc gtc ctg 168 Met Leu Phe Asn Ser Val LeuArg Gln Pro Gln Leu Gly Val Leu 1 5 10 15 aga aat gga tgg tct tca caatac cct ctt caa tcc ctt ctg act ggt 216 Arg Asn Gly Trp Ser Ser Gln TyrPro Leu Gln Ser Leu Leu Thr Gly 20 25 30 tat cag tgc agt ggt aat gat gaacac act tct tat gga gaa aca gga 264 Tyr Gln Cys Ser Gly Asn Asp Glu HisThr Ser Tyr Gly Glu Thr Gly 35 40 45 gtc cca gtt cct cct ttt gga tgt accttc tct tct gct ccc aat atg 312 Val Pro Val Pro Pro Phe Gly Cys Thr PheSer Ser Ala Pro Asn Met 50 55 60 gaa cat gta cta gca gtt gcc aat gaa gaaggc ttt gtt cga ttg tat 360 Glu His Val Leu Ala Val Ala Asn Glu Glu GlyPhe Val Arg Leu Tyr 65 70 75 aac aca gaa tca caa agt ttc aga aag aag tgcttc aaa gaa tgg atg 408 Asn Thr Glu Ser Gln Ser Phe Arg Lys Lys Cys PheLys Glu Trp Met 80 85 90 95 gct cac tgg aat gcc gtc ttt gac ctg gcc tgggtt cct ggt gaa ctt 456 Ala His Trp Asn Ala Val Phe Asp Leu Ala Trp ValPro Gly Glu Leu 100 105 110 aaa ctt gtt aca gca gca ggt gat caa aca gccaaa ttt tgg gac gta 504 Lys Leu Val Thr Ala Ala Gly Asp Gln Thr Ala LysPhe Trp Asp Val 115 120 125 aaa gct ggt gag ctg att gga aca tgc aaa ggtcat caa tgc agc ctc 552 Lys Ala Gly Glu Leu Ile Gly Thr Cys Lys Gly HisGln Cys Ser Leu 130 135 140 aag tca gtt gcc ttt tct aag ttt gag aaa gctgta ttc tgt acg ggt 600 Lys Ser Val Ala Phe Ser Lys Phe Glu Lys Ala ValPhe Cys Thr Gly 145 150 155 gga aga gat ggc aac att atg gtc tgg gat accagg tgc aac aaa aaa 648 Gly Arg Asp Gly Asn Ile Met Val Trp Asp Thr ArgCys Asn Lys Lys 160 165 170 175 gat ggg ttt tat agg caa gtg aat caa atcagt gga gct cac aat acc 696 Asp Gly Phe Tyr Arg Gln Val Asn Gln Ile SerGly Ala His Asn Thr 180 185 190 tca gac aag caa acc cct tca aaa ccc aagaag aaa cag aat tca aaa 744 Ser Asp Lys Gln Thr Pro Ser Lys Pro Lys LysLys Gln Asn Ser Lys 195 200 205 gga ctt gct cct tct gtg gat ttc cag caaagt gtt act gtg gtc ctc 792 Gly Leu Ala Pro Ser Val Asp Phe Gln Gln SerVal Thr Val Val Leu 210 215 220 ttt caa gac gag aat acc tta gtc tca gcagga gct gtg gat ggg ata 840 Phe Gln Asp Glu Asn Thr Leu Val Ser Ala GlyAla Val Asp Gly Ile 225 230 235 atc aaa gta tgg gat tta cgt aag aat tatact gct tat cga caa gaa 888 Ile Lys Val Trp Asp Leu Arg Lys Asn Tyr ThrAla Tyr Arg Gln Glu 240 245 250 255 ccc ata gca tcc aag tct ttc ctg taccca ggt agc agc act cga aaa 936 Pro Ile Ala Ser Lys Ser Phe Leu Tyr ProGly Ser Ser Thr Arg Lys 260 265 270 ctt gga tat tca agt ctg att ttg gattcc act ggc tct act tta ttt 984 Leu Gly Tyr Ser Ser Leu Ile Leu Asp SerThr Gly Ser Thr Leu Phe 275 280 285 gct aat tgc aca gac gat aac atc tacatg ttt aat atg act ggg ttg 1032 Ala Asn Cys Thr Asp Asp Asn Ile Tyr MetPhe Asn Met Thr Gly Leu 290 295 300 aag act tct cca gtg gct att ttc aatgga cac cag aac tct acc ttt 1080 Lys Thr Ser Pro Val Ala Ile Phe Asn GlyHis Gln Asn Ser Thr Phe 305 310 315 tat gta aaa tcc agc ctt agt cca gatgac cag ttt tta gtc agt ggc 1128 Tyr Val Lys Ser Ser Leu Ser Pro Asp AspGln Phe Leu Val Ser Gly 320 325 330 335 tca agt gat gaa gct gcc tac atatgg aag gtc tcc aca ccc tgg caa 1176 Ser Ser Asp Glu Ala Ala Tyr Ile TrpLys Val Ser Thr Pro Trp Gln 340 345 350 cct cct act gtg ctc ctg ggt cattct caa gag gtc acg tct gtg tgc 1224 Pro Pro Thr Val Leu Leu Gly His SerGln Glu Val Thr Ser Val Cys 355 360 365 tgg tgt cca tct gac ttc aca aagatt gct acc tgt tct gat gac aat 1272 Trp Cys Pro Ser Asp Phe Thr Lys IleAla Thr Cys Ser Asp Asp Asn 370 375 380 aca cta aaa atc tgg cgc ttg aataga ggc tta gag gag aaa cca gga 1320 Thr Leu Lys Ile Trp Arg Leu Asn ArgGly Leu Glu Glu Lys Pro Gly 385 390 395 ggt gat aaa ctt tcc acg gtg ggttgg gcc tct cag aag aaa aaa gag 1368 Gly Asp Lys Leu Ser Thr Val Gly TrpAla Ser Gln Lys Lys Lys Glu 400 405 410 415 tca aga cct ggc cta gta acagta acg agt agc cag agt act cct gcc 1416 Ser Arg Pro Gly Leu Val Thr ValThr Ser Ser Gln Ser Thr Pro Ala 420 425 430 aaa gcc ccc agg gta aag tgcaat cca tcc aat tct tcc ccg tca tcc 1464 Lys Ala Pro Arg Val Lys Cys AsnPro Ser Asn Ser Ser Pro Ser Ser 435 440 445 gca gct tgt gcc cca agc tgtgct gga gac ctc cct ctt cct tca aat 1512 Ala Ala Cys Ala Pro Ser Cys AlaGly Asp Leu Pro Leu Pro Ser Asn 450 455 460 act cct acg ttc tct att aaaacc tct cct gcc aag gcc cgg tct ccc 1560 Thr Pro Thr Phe Ser Ile Lys ThrSer Pro Ala Lys Ala Arg Ser Pro 465 470 475 atc aac aga aga ggc tct gtctcc tcc gtc tct ccc aag cca cct tca 1608 Ile Asn Arg Arg Gly Ser Val SerSer Val Ser Pro Lys Pro Pro Ser 480 485 490 495 tct ttc aag atg tcg attaga aac tgg gtg acc cga aca cct tcc tca 1656 Ser Phe Lys Met Ser Ile ArgAsn Trp Val Thr Arg Thr Pro Ser Ser 500 505 510 tca cca ccc atc act ccacct gct tcg gag acc aag atc atg tct ccg 1704 Ser Pro Pro Ile Thr Pro ProAla Ser Glu Thr Lys Ile Met Ser Pro 515 520 525 aga aaa gcc ctt att cctgtg agc cag aag tca tcc caa gca gag gct 1752 Arg Lys Ala Leu Ile Pro ValSer Gln Lys Ser Ser Gln Ala Glu Ala 530 535 540 tgc tct gag tct aga aataga gta aag agg agg cta gac tca agc tgt 1800 Cys Ser Glu Ser Arg Asn ArgVal Lys Arg Arg Leu Asp Ser Ser Cys 545 550 555 ctg gag agt gtg aaa caaaag tgt gtg aag agt tgt aac tgt gtg act 1848 Leu Glu Ser Val Lys Gln LysCys Val Lys Ser Cys Asn Cys Val Thr 560 565 570 575 gag ctt gat ggc caagtt gaa aat ctt cat ttg gat ctg tgc tgc ctt 1896 Glu Leu Asp Gly Gln ValGlu Asn Leu His Leu Asp Leu Cys Cys Leu 580 585 590 gct ggt aac cag gaagac ctt agt aag gac tct cta ggt cct acc aaa 1944 Ala Gly Asn Gln Glu AspLeu Ser Lys Asp Ser Leu Gly Pro Thr Lys 595 600 605 tca agc aaa att gaagga gct ggt acc agt atc tca gag cct ccg tct 1992 Ser Ser Lys Ile Glu GlyAla Gly Thr Ser Ile Ser Glu Pro Pro Ser 610 615 620 cct atc agt ccg tatgct tca gaa agc tgt gga acg cta cct ctt cct 2040 Pro Ile Ser Pro Tyr AlaSer Glu Ser Cys Gly Thr Leu Pro Leu Pro 625 630 635 ttg aga cct tgt ggagaa ggg tct gaa atg gta ggc aaa gag aat agt 2088 Leu Arg Pro Cys Gly GluGly Ser Glu Met Val Gly Lys Glu Asn Ser 640 645 650 655 tcc cca gag aataaa aac tgg ttg ttg gcc atg gca gcc aaa cgg aag 2136 Ser Pro Glu Asn LysAsn Trp Leu Leu Ala Met Ala Ala Lys Arg Lys 660 665 670 gct gag aat ccatct cca cga agt ccg tca tcc cag aca ccc aat tcc 2184 Ala Glu Asn Pro SerPro Arg Ser Pro Ser Ser Gln Thr Pro Asn Ser 675 680 685 agg aga cag agcgga aag aca ttg cca agc ccg gtc acc atc acg ccc 2232 Arg Arg Gln Ser GlyLys Thr Leu Pro Ser Pro Val Thr Ile Thr Pro 690 695 700 agc tcc atg aggaaa atc tgc aca tac ttc cat aga aag tcc cag gag 2280 Ser Ser Met Arg LysIle Cys Thr Tyr Phe His Arg Lys Ser Gln Glu 705 710 715 gac ttc tgt ggtcct gaa cac tca aca gaa tta tagattctaa tctgagtgag 2333 Asp Phe Cys GlyPro Glu His Ser Thr Glu Leu 720 725 730 ttactgagct ttggtccact aaaacaagctgagctttggt ccactaaaac aagatgaaaa 2393 atacaagagt gactctataa ctctggtctttaagaaagct gccttttcat ttttagacaa 2453 aatcttttca acgctgaaat gtacctaatctggttctact accataatgt atatgcagct 2513 tcccgaggat gaatgctgtg tttaaatttcataaagtaaa tttgtcactc tagcattttg 2573 aatgaatagt cttcactttt taaattattcatcttctcta taataatgac atcccagttc 2633 atggaggcaa aaaacaagtt tcttgttatcctgaaacttt ctatgctcag tggaaagtat 2693 ctgccagcca cagcatgagg cctgtgaaggctgactgaga aatcctctgc tgaagacccc 2753 tggttctgtt ctgcctccaa catgtataattttatttgaa atacataatc ttttcactat 2813 gaaaaaaaaa aaaaaaaa 2831 2 730 PRTHomo sapiens 2 Met Leu Phe Asn Ser Val Leu Arg Gln Pro Gln Leu Gly ValLeu Arg 1 5 10 15 Asn Gly Trp Ser Ser Gln Tyr Pro Leu Gln Ser Leu LeuThr Gly Tyr 20 25 30 Gln Cys Ser Gly Asn Asp Glu His Thr Ser Tyr Gly GluThr Gly Val 35 40 45 Pro Val Pro Pro Phe Gly Cys Thr Phe Ser Ser Ala ProAsn Met Glu 50 55 60 His Val Leu Ala Val Ala Asn Glu Glu Gly Phe Val ArgLeu Tyr Asn 65 70 75 80 Thr Glu Ser Gln Ser Phe Arg Lys Lys Cys Phe LysGlu Trp Met Ala 85 90 95 His Trp Asn Ala Val Phe Asp Leu Ala Trp Val ProGly Glu Leu Lys 100 105 110 Leu Val Thr Ala Ala Gly Asp Gln Thr Ala LysPhe Trp Asp Val Lys 115 120 125 Ala Gly Glu Leu Ile Gly Thr Cys Lys GlyHis Gln Cys Ser Leu Lys 130 135 140 Ser Val Ala Phe Ser Lys Phe Glu LysAla Val Phe Cys Thr Gly Gly 145 150 155 160 Arg Asp Gly Asn Ile Met ValTrp Asp Thr Arg Cys Asn Lys Lys Asp 165 170 175 Gly Phe Tyr Arg Gln ValAsn Gln Ile Ser Gly Ala His Asn Thr Ser 180 185 190 Asp Lys Gln Thr ProSer Lys Pro Lys Lys Lys Gln Asn Ser Lys Gly 195 200 205 Leu Ala Pro SerVal Asp Phe Gln Gln Ser Val Thr Val Val Leu Phe 210 215 220 Gln Asp GluAsn Thr Leu Val Ser Ala Gly Ala Val Asp Gly Ile Ile 225 230 235 240 LysVal Trp Asp Leu Arg Lys Asn Tyr Thr Ala Tyr Arg Gln Glu Pro 245 250 255Ile Ala Ser Lys Ser Phe Leu Tyr Pro Gly Ser Ser Thr Arg Lys Leu 260 265270 Gly Tyr Ser Ser Leu Ile Leu Asp Ser Thr Gly Ser Thr Leu Phe Ala 275280 285 Asn Cys Thr Asp Asp Asn Ile Tyr Met Phe Asn Met Thr Gly Leu Lys290 295 300 Thr Ser Pro Val Ala Ile Phe Asn Gly His Gln Asn Ser Thr PheTyr 305 310 315 320 Val Lys Ser Ser Leu Ser Pro Asp Asp Gln Phe Leu ValSer Gly Ser 325 330 335 Ser Asp Glu Ala Ala Tyr Ile Trp Lys Val Ser ThrPro Trp Gln Pro 340 345 350 Pro Thr Val Leu Leu Gly His Ser Gln Glu ValThr Ser Val Cys Trp 355 360 365 Cys Pro Ser Asp Phe Thr Lys Ile Ala ThrCys Ser Asp Asp Asn Thr 370 375 380 Leu Lys Ile Trp Arg Leu Asn Arg GlyLeu Glu Glu Lys Pro Gly Gly 385 390 395 400 Asp Lys Leu Ser Thr Val GlyTrp Ala Ser Gln Lys Lys Lys Glu Ser 405 410 415 Arg Pro Gly Leu Val ThrVal Thr Ser Ser Gln Ser Thr Pro Ala Lys 420 425 430 Ala Pro Arg Val LysCys Asn Pro Ser Asn Ser Ser Pro Ser Ser Ala 435 440 445 Ala Cys Ala ProSer Cys Ala Gly Asp Leu Pro Leu Pro Ser Asn Thr 450 455 460 Pro Thr PheSer Ile Lys Thr Ser Pro Ala Lys Ala Arg Ser Pro Ile 465 470 475 480 AsnArg Arg Gly Ser Val Ser Ser Val Ser Pro Lys Pro Pro Ser Ser 485 490 495Phe Lys Met Ser Ile Arg Asn Trp Val Thr Arg Thr Pro Ser Ser Ser 500 505510 Pro Pro Ile Thr Pro Pro Ala Ser Glu Thr Lys Ile Met Ser Pro Arg 515520 525 Lys Ala Leu Ile Pro Val Ser Gln Lys Ser Ser Gln Ala Glu Ala Cys530 535 540 Ser Glu Ser Arg Asn Arg Val Lys Arg Arg Leu Asp Ser Ser CysLeu 545 550 555 560 Glu Ser Val Lys Gln Lys Cys Val Lys Ser Cys Asn CysVal Thr Glu 565 570 575 Leu Asp Gly Gln Val Glu Asn Leu His Leu Asp LeuCys Cys Leu Ala 580 585 590 Gly Asn Gln Glu Asp Leu Ser Lys Asp Ser LeuGly Pro Thr Lys Ser 595 600 605 Ser Lys Ile Glu Gly Ala Gly Thr Ser IleSer Glu Pro Pro Ser Pro 610 615 620 Ile Ser Pro Tyr Ala Ser Glu Ser CysGly Thr Leu Pro Leu Pro Leu 625 630 635 640 Arg Pro Cys Gly Glu Gly SerGlu Met Val Gly Lys Glu Asn Ser Ser 645 650 655 Pro Glu Asn Lys Asn TrpLeu Leu Ala Met Ala Ala Lys Arg Lys Ala 660 665 670 Glu Asn Pro Ser ProArg Ser Pro Ser Ser Gln Thr Pro Asn Ser Arg 675 680 685 Arg Gln Ser GlyLys Thr Leu Pro Ser Pro Val Thr Ile Thr Pro Ser 690 695 700 Ser Met ArgLys Ile Cys Thr Tyr Phe His Arg Lys Ser Gln Glu Asp 705 710 715 720 PheCys Gly Pro Glu His Ser Thr Glu Leu 725 730 3 83 DNA Homo sapiens 3tttgagaaag ctgtattctg tacgggtgga agagatggca acattatggt ctgggatacc 60aggtgcaaca aaaaagatgg gtt 83

1. A gene having the sequence of SEQ ID NO:
 1. 2. An expression productencoded by a gene according to claim
 1. 3. An expression productaccording to claim 2, having the sequence of SEQ ID NO:
 2. 4. Anexpression product according to claim 3, wherein said expression productis a mitogen.
 5. An expression product according to claim 3, whereinsaid, expression product is a morphogen.
 6. An expression productaccording to claim 2, wherein said expression product comprises aprotein.
 7. An expression product according to claim 2, wherein saidexpression product affects the identity of stem cell progenitor cells.8. An expression product according to claim 2 which affects embryonicdevelopment.
 9. An expression product according to claim 8, whichaffects the development of embryonic brain, lung, liver and kidneytissues.
 10. An expression product according to claim 2, which affectscancer cell differentiation.
 11. An expression product according toclaim 2, for use in the treatment or prevention of Ushers Disease.
 12. Arecombinant DNA construct comprising operatively linked in sequence inthe 5′ to 3′ direction: a) a promoter region that directs thetranscription of a gene; b) a DNA coding sequence encoding an RNAsequence encoding an expression product according to claim 2; and c) a3′ non-translated region.
 13. A recombinant DNA construct according toclaim 12, the DNA coding sequence having the sequence of SEQ ID NO: 1.14. A cell transformed or transfected with a recombinant DNA constructaccording to claim
 12. 15. A method of treating or preventing diseasesassociated with the expression of a gene comprising administering to apatient an expression product according to claim
 2. 16. A chromosomemarker for band q 32.1-32.2 having the sequence of SEQ ID NO:2.